Rapid Detection and Identification of Mycotoxigenic Fungi And

toxins

Article Rapid Detection and Identiﬁcation of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain

Sudharsan Sadhasivam 1, Malka Britzi 2, Varda Zakin 1, Moshe Kostyukovsky 1, Anatoly Trostanetsky 1, Elazar Quinn 1 and Edward Sionov 1,*

1 Department of Food Quality and Safety, Institute for Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center, Rishon LeZion 7528809, Israel; [email protected] (S.S.); [email protected] (V.Z.); [email protected] (M.K.); [email protected] (A.T.); [email protected] (E.Q.) 2 National Residue Control Laboratory, Kimron Veterinary Institute, Bet Dagan 50250, Israel; [email protected] * Correspondence: [email protected]; Tel.: +972-3-9683693

Academic Editor: Laura Anfossi Received: 24 August 2017; Accepted: 20 September 2017; Published: 25 September 2017

Abstract: This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for Fusarium and Aspergillus species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B1, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains.

Keywords: toxigenic fungi; aﬂatoxins; fumonisins; deoxynivalenol; wheat grains; multiplex PCR; LC/MS/MS

1. Introduction Wheat grain and associated by-products are important sources of energy and protein for humans and all classes of farm animals. When grains are colonized by moulds there is a significant risk of contamination with mycotoxins, which are toxic chemical products, formed as secondary metabolites by these fungi. Many species of Fusarium, Aspergillus, Penicillium, and Alternaria are not only recognised as plant pathogens but are also sources of important mycotoxins of concern in relation to animal and human health [1]. Traditionally, toxigenic fungi in crops have been divided into two distinct classes: ‘field fungi’ (or plant pathogens), which invade and produce their toxins before harvest; and ‘storage’ (or saprophytic) fungi, which become a problem after harvest. However, the original source of both of these classes of fungi is in the field [2]. Moreover, some fungi might belong to both classes and colonize grains before and after harvest. Aspergillus flavus, for example, is associated with

Toxins 2017, 9, 302; doi:10.3390/toxins9100302 www.mdpi.com/journal/toxins Toxins 2017, 9, 302 2 of 17

Aspergillus infections of grain crops in the field; it also contaminates stored grains when prevailing abiotic factors (such as temperature and water activity) are favourable [3,4]. Interactions between environmental stress factors, such as water activity and temperature, may have an influence on growth, expression of biosynthetic regulatory genes and mycotoxin production by mycotoxigenic fungal species [5,6]. Good postharvest practices avoiding high temperatures and rapid drying of grain can avoid a decrease in the grain quality and reduce the health risk due to mould growth and potential toxin contamination [6]. Poor postharvest management can lead to rapid deterioration in quality, with severe decreases in germinability and nutritional value of stored grain, possibly accompanied by undesirable fungal contamination and, consequently, toxin production [7]. The most frequently detected mycotoxins in wheat grain are deoxynivalenol (DON), fumonisins (Fs), and zearalenone (ZEN), produced by Fusarium species, and aflatoxins (AFs) and ochratoxin A (OTA) produced by Aspergillus and Penicillium species, respectively [8]. Due to their wide range of physical and chemical properties, mycotoxins are stable chemical compounds which cannot be destroyed during most food processing operations. The Food and Agricultural Organization (FAO) of the United Nations estimated that each year approximately 25% of the world’s crops are contaminated by mycotoxins, which cause annual losses of around one billion metric tons of food products. Timely assessment of these contaminants and identification of the main toxicogenic fungal species are important, not only for assessing food quality, but also for the development of control strategies for ensuring food safety [9]. Surveillance and monitoring for mycotoxigenic fungi and mycotoxins becomes critical for maintaining a high quality of grains and grain products in indoor storage facilities. Molecular approaches, such as PCR, can serve as good alternatives to conventional methods for detection of mycotoxigenic fungi. Multiplex PCR assays that simultaneously amplify numbers of species-specific genes, and/or structural or regulatory genes involved in mycotoxin biosynthesis pathways have been successfully applied to the detection of mycotoxigenic fungi in a variety of foods and feeds [10–13]. However, there have been only a few examples of differentiation of mycotoxin-producing fungi in wheat grains by means of multiplex PCR. Wang and co-workers developed a multiplex PCR assay for simultaneous detection of genes involved in biosynthesis of type B trichothecene mycotoxins that are found in wheat grain [14], with emphasis on the genetic identification of certain mycotoxin chemotypes of Fusarium species. Further work is now needed in order to develop a sensitive PCR assays for detection of a wide range of mycotoxigenic fungi in cereal grains. Traditionally, mycotoxin analyses are mainly performed by means of high-performance liquid chromatography after clean-up in a solid phase extraction/immunoaffinity column [15,16]. These methods normally enable the determination of only single classes of mycotoxins, including a limited number of target analytes, increasing the cost and time of the analysis due to labour-intensive sample preparation. In order to improve the sensitivity of mycotoxin detection and to reduce the cost of the analyses it would be preferable to determine as many mycotoxins as possible by routine analysis in different types of matrices in one single extraction. Furthermore, multi-mycotoxin determination methods are required because of the co-occurrence of several mycotoxins in various commodities [17]. The complexity of the wheat grain matrix, as well as the wide range of physical and chemical properties of mycotoxins, necessitate selective and sensitive detection techniques for co-occurring toxins. Liquid chromatography/mass spectrometry (LC/MS), and particularly LC coupled to tandem mass spectrometry (LC/MS/MS) have become very popular in recent years for mycotoxin analysis. However, a multi-toxin sample extraction and preparation can be challenging due to matrix effects and chemical diversity of the analytes. Solid phase extraction methodology has been developed for analysis of a number of mycotoxins in foodstuffs [18–20]. The major drawback of this procedure is that it is time consuming, especially when large numbers of samples need to be analysed [21]. Immunoaffinity columns, for clean-up purposes, have become increasingly popular in recent years due to the high degree of selectivity and specificity [22–24]. The fact that these columns are only used once and their relative high costs are major disadvantages. The use of various modifications Toxins 2017, 9, 302 3 of 17 of the QuEChERS methodology (quick, easy, cheap, effective, rugged, and safe approach) has also been recently introduced in multi-mycotoxin analysis [25]; however, the efficiency of the method for eliminating matrix effects has yet to be determined. In the current study we developed a new low-cost, rapid, and simple extraction and analysis method for multi-toxin detection in stored wheat grains. Considering the chemical diversity of the analytes, crude sample extracts were injected into the LC/MS/MS system without further purification. The method was successfully applied for rapid and simultaneous determination of 10 mycotoxins in stored wheat grains. To the best of our knowledge, this is the first report on evaluation and optimization of a reliable multiplex PCR assay and LC/MS/MS multi-method for simultaneous detection and accurate determination of the critically important mycotoxigenic fungi and mycotoxins, respectively, in wheat grains collected from storage warehouses in Israel. This combined approach will enable to evaluate the mycotoxicological risk of stored wheat grains, to minimize economic losses and reduce the hazard to animal and human health.

2. Results and Discussion

2.1. Specificity and Sensitivity of Multiplex PCR In this study, a multiplex PCR assay was used to facilitate simultaneous detection of a wide range of potentially mycotoxin-producing fungi in wheat grain storage warehouses in Israel. Five sets of four or five species-specific primer pairs were assembled for the molecular identification of the fungal species most frequently found in stored wheat grain [26]. They comprised seven Aspergillus spp., nine Fusarium spp., and five Penicillium spp. (Table1).

Table 1. Sets of species-speciﬁc primers used in this study 1.

Annealing Set No. Species DNA/Gene Target Amplicon Size (bp) Temperature A. tubingensis caM 505 A. parasiticus caM 430 I A. carbonarius caM 371 60 ◦C A. fumigatus pep 250 A. ﬂavus pepO 200 F. culmorum RAPD 2 marker 570 F. graminearum RAPD marker 400–500 II F. sporotrichioides tri13 332 55 ◦C F. poae RAPD marker 220 F. avenaceum RAPD marker 920 F. verticillioides caM 578 III A. niger caM 357 55 ◦C F. solani AFLP 175 F. proliferatum caM 585 P. expansum IDH 480 IV F. oxysporum ITS 340 55 ◦C P. digitatum Cyp51 250 P. verrucosum Otanps 750 P. paneum IDH 482 V A. terreus Topoisomerase II 386 60 ◦C P. roqueforti ITS1-5.8S-ITS2 300 1 See Table S1 for detailed description of the primers used in the study; 2 RAPD: random ampliﬁed polymorphic DNA.

The specificity of all the species-specific primers was assessed by performing multiplex PCR on the genomic DNA of standard fungal isolates, which resulted in complete amplification of all target genes (Figure1A). In addition, three sets of four to six primers were assembled, to amplify Toxins 2017, 9, 302 4 of 17 the genes associated with mycotoxin biosynthesis, in order to detect mycotoxigenic fungi (Table2). The annealing temperature of each primer group for amplification of the target genes was set by using temperature gradient conditions. The specificity of the developed multiplex PCR assay was confirmed; itToxins provided 2017, 9 good, 302 discrimination among the tested species, as well as between mycotoxin-producing4 of 16 and non-producing strains. In addition, none of the primers showed cross-reactions with the species temperature gradient conditions. The specificity of the developed multiplex PCR assay was amplified by the respective other primers. confirmed; it provided good discrimination among the tested species, as well as between mycotoxin‐producingTable and 2. Primer non‐producing sets for genes strains. involved In in addition, mycotoxin biosynthesisnone of the1. primers showed cross‐reactions with the species amplified by the respective other primers. Amplicon Size Annealing Set No. Mycotoxin Gene Target Table 2. Primer sets for genes involved in mycotoxin biosynthesis(bp) 1. Temperature

Set No. Mycotoxin Gene Target avfAAmplicon Size (bp) 950Annealing Temperature aﬂR1 798 VI Aﬂatoxins avfA 950 ver1 452 58 ◦C aflR1 798 VI Aflatoxins nor1 397 ver1 452 58 °C fum13 988

nor1 fum1 397 798 VII Fumonisin/Trichothecene/Zearalenonefum13 tri6 988 546 55 ◦C fum1 tri5 798 450 Fumonisin/Trichothecene/ VII tri6 pks13 546 351 55 °C Zearalenone tri5 450 VIII Ochratoxin A pks13 otanps 351 788 58 ◦C 58 °C VIII Ochratoxin A otanps 788 1 See Table S1 for detailed description of the primers used in the study. 1 See Table S1 for detailed description of the primers used in the study.

FigureFigure 1. 1. SpecificitySpecificity and and sensitivity sensitivity of ofmultiplex multiplex PCR PCR assays assays using using species species specific specific primers. primers. (A) (ASpecificity) Specificity test. test. Lanes: Lanes: M‐100 M-100 bp DNA bp DNA ladder; ladder; 1: primer 1: primer set I; 2: set primer I; 2: primer set II; 3: set primer II; 3: set primer III; 4: set III;primer 4: primer set IV; set 5: IV; primer 5: primer set V; setand V; (B and) sensitivity (B) sensitivity test. Lanes: test. 1: Lanes: negative 1: negative control; 2: control; positive 2: control positive control(wheat (wheat sample sample inoculated inoculated by A. parasiticus by A. parasiticus NRRL 6111,NRRL A.fumigatus 6111, A. fumigatus NRRL 62427,NRRL A. 62427, carbonariusA. carbonarius NRRL NRRL368 and 368 andincubated incubated for 48 for hrs); 48 hrs); 3–5 3–5(represent (represent samples samples inoculated inoculated by byA. A.parasiticus parasiticus NRRLNRRL 6111, 6111, 4 5 A.fumigatusA.fumigatusNRRL NRRL 62427, 62427,A. carbonariusA. carbonariusNRRL NRRL 368 without 368 without incubation): incubation): 3: 104 spores/g;3: 10 spores/g; 4: 105 spores/g; 4: 10 6 andspores/g; 5: 106 spores/g. and 5: 10 spores/g.

The PCR‐based detection of microorganisms in food and feed, as well as in biological samples, is alwaysThe PCR-based challenging detection because of microorganismsthe variety of species in food that andneed feed, to be as determined, well as in or biological the presence samples, of issubstances always challenging that inhibit because the PCR of theor reduce variety its of amplification species that need efficiency, to be determined,either of which or the can presence cause ofcomplete substances reaction that inhibitfailure, theleading PCR to or false reduce negative its amplification results or reduced efficiency, sensitivity either of of specific which detection can cause completeof the mycotoxin reaction failure,‐producing leading moulds to false [27]. negative However, results in the or reduced present sensitivitystudy, the ofnewly specific‐developed detection ofassays the mycotoxin-producing have advantages over moulds previously [27‐].reported However, multiplex in the PCRs present in terms study, of the the newly-developed broad range of assaysmycotoxigenic have advantages fungi detected over previously-reported in a few single assays, multiplex which could PCRs detect in terms all target of the fungal broad species range of mycotoxigenicwith higher specificity fungi detected (Figure in 1A). a few To singledetermine assays, the whichminimum could amount detect of all fungal target template fungal species necessary with higherto obtain specificity visible (Figure amplification1A). To products, determine the the multiplex minimum PCR amount assays of were fungal carried template out necessarywith serial to dilutions of fungal genomic DNA. The detection limit for purified genomic DNA to produce a visible band on an ethidium bromide‐stained agarose gel was determined to be 100 pg for all strains tested. When the sensitivity of the multiplex PCR was evaluated by artificial inoculation of wheat grain samples with known amounts of Aspergillus spp. conidia, the detection limit for DNA of each species, extracted directly from grains, was 1 × 104 spores/g in samples with no incubation (Figure

Toxins 2017, 9, 302 5 of 17 obtain visible amplification products, the multiplex PCR assays were carried out with serial dilutions of fungal genomic DNA. The detection limit for purified genomic DNA to produce a visible band on an ethidium bromide-stained agarose gel was determined to be 100 pg for all strains tested. WhenToxins 2017 the, 9 sensitivity, 302 of the multiplex PCR was evaluated by artificial inoculation of wheat grain5 of 16 samples with known amounts of Aspergillus spp. conidia, the detection limit for DNA of each species, extracted1B). The sensitivity directly from of the grains, PCR wasreaction 1 × might104 spores/g be influenced in samples by various with no components incubation of (Figure biological1B). Thematrices, sensitivity such ofas thefats, PCR or phenolic reaction mightand polysaccharide be influenced bycompounds, various components which can of reduce biological the purity matrices, of suchthe extracted as fats, or DNA phenolic [10,28,29]. and polysaccharide In some previous compounds, studies, which an enrichment can reduce thestep purity was performed of the extracted that DNAinvolved [10, 28incubation,29]. In some of previouscontaminated studies, food an enrichmentsamples for step a certain was performed period of that time, involved to improve incubation the ofsensitivity contaminated of fungal food‐ samplesspecific PCR for a certainassays period[8,12,30]. of time,Nevertheless, to improve in the the sensitivity present study, of fungal-specific the higher PCRefficacy assays of the [8, 12assay,30]. was Nevertheless, achieved by in use the presentof an efficient study, DNA the higher extraction efficacy method of the and assay optimized was achieved PCR byconditions, use of an and efficient it resulted DNA in extraction an improvement method andof at optimized least one log PCR of conditions, sensitivity, and compared it resulted with in that an improvementreported in previous of at least studies one log [10,31]. of sensitivity, compared with that reported in previous studies [10,31].

2.2.2.2. Application to Stored WheatWheat GrainGrain SamplesSamples InIn lightlight ofof thethe highhigh importanceimportance ofof wheatwheat graingrain storagestorage withinwithin thethe marketing,marketing, distribution,distribution, andand foodfood security system, the the high high quality quality and and safety safety of of this this product product need need to to be be ensured. ensured. In Inorder order to tocharacterize characterize mycotoxin mycotoxin-producing‐producing fungi fungi by by multiplex multiplex PCR, PCR, sets sets of of the the primers primers directed directed to thethe structuralstructural and regulatoryregulatory genesgenes involvedinvolved inin biosynthesisbiosynthesis of aflatoxinsaflatoxins ((aflR1aflR1,, nor1, avfA, ver1), FusariumFusarium toxinstoxins ((fum1fum1,, fum13fum13, tri5, tri6, zea), and OTA (pks, otanps) were testedtested by usingusing DNADNA extractedextracted from variousvarious species of Aspergillus, Fusarium, and Penicillium, respectively (Figure 22A–C).A–C). TheThe obtainedobtained resultsresults clearlyclearly showshow thethe presencepresence ofof specificspecific fragmentsfragments amplifiedamplified fromfrom DNADNA ofof isolatesisolates withwith potentialpotential mycotoxin-producingmycotoxin‐producing abilities.abilities. SomeSome AspergillusAspergillus strainsstrains ((A.A. flavusflavus NRRLNRRL 3518,3518, A.A. flavusflavus SS2,SS2, A.A. carbonarius NRRL 368, and A. ochraceus NRRL 35018) did not show amplificationamplification in any target genesgenes (Figure(Figure2 A,C).2A,C). The The probability probability of of a particulara particular toxin toxin being being produced produced can can be be predicted predicted according according to theto the presence presence or absence or absence of an of amplification an amplification product, product, but the but effective the effective biosynthesis biosynthesis of the toxin of the remains toxin toremains be confirmed to be confirmed by analytical by analytical chemistry chemistry analysis [analysis32,33]. [32,33].

FigureFigure 2. SpecificitySpecificity of of multiplex multiplex PCR PCR assays assays using using sets setsof primers of primers for mycotoxin for mycotoxin biosynthetic biosynthetic genes. genes.(A) Primer (A) set Primer VI. Lanes: set VI. M: Lanes: 1 kb DNA M: 1 ladder; kb DNA 1: A. ladder; parasiticus 1: A. NRRL parasiticus 6111; 2:NRRL A. flavus 6111; NRRL 2: A. 3518; flavus 3: NRRLA. flavus 3518; SS1; 3: 4:A. A. flavus flavusSS1; SS2; 4: A.(B) flavus primerSS2; set ( BVII.) primer Lanes: set 1: VII. F. Lanes:verticillioides 1: F. verticillioidesNRRL 25457;NRRL 2: F. 25457;sporotrichioides 2: F. sporotrichioides NRRL 3299;NRRL 3: F. 3299; culmorum 3: F. culmorum NRRL 13320;NRRL 4: 13320; F. graminearum 4: F. graminearum NRRL NRRL3376; 3376;5: F. 5:verticillioidesF. verticillioides SS4;SS4; 6: F. 6: verticillioidesF. verticillioides SS5;SS5; and and (C) (primerC) primer set setVIII. VIII. Lanes: Lanes: 1: P. 1: verrucosumP. verrucosum NRRLNRRL 965; 965; 2: 2:P. P.viridicatum viridicatum NRRLNRRL 5571; 5571; 3: 3: A.A. carbonarius carbonarius NRRLNRRL 368; 368; and and 4: 4: A.A. ochraceus ochraceus NRRLNRRL 35018. 35018.

Primer sets for genes related to aflatoxin and Fusarium toxin biosynthesis were also tested on Primer sets for genes related to aflatoxin and Fusarium toxin biosynthesis were also tested on DNA samples obtained from wheat grain artificially infected with A. parasiticus NRRL 6111 (Figure DNA samples obtained from wheat grain artificially infected with A. parasiticus NRRL 6111 (Figure3A) 3A) and Fusarium spp. (Figure 3B). These primers enabled us to identify potential aflatoxigenic, and Fusarium spp. (Figure3B). These primers enabled us to identify potential aflatoxigenic, fumonisin- fumonisin‐ and trichothecene‐producing isolates in wheat grains. Primer sets for genes related to aflatoxin and Fusarium toxin biosynthesis were also tested on DNA samples obtained from wheat grain artificially infected with A. parasiticus NRRL 6111 (Figure 3A) and Fusarium spp. (Figure 3B). These primers enabled us to identify potential aflatoxigenic, fumonisin‐, and trichothecene‐producing isolates in wheat grains.

Toxins 2017, 9, 302 6 of 17 and trichothecene-producing isolates in wheat grains. Primer sets for genes related to aflatoxin and Fusarium toxin biosynthesis were also tested on DNA samples obtained from wheat grain artificially infected with A. parasiticus NRRL 6111 (Figure3A) and Fusarium spp. (Figure3B). These primers enabled us to identify potential aflatoxigenic, fumonisin-, and trichothecene-producing isolates in wheatToxins 2017 grains., 9, 302 6 of 16

FigureFigure 3. 3. SpecificitySpecificity of of multiplex multiplex PCR PCR assays assays in in artificially artificially contaminated contaminated wheat wheat grain grain samples. samples. (A) (APrimer) Primer set set VI. VI. Lanes: Lanes: M: M: 1 1kb kb DNA DNA ladder; ladder; 1: 1: negative negative control (no(no inoculation);inoculation); 2:2: wheatwheat sample sample artificiallyartificially inoculated inoculated with withA. A. parasiticus parasiticusNRRL NRRL 6111; 6111; and and ( B(B)) primer primer set set VII. VII. Lanes: Lanes: 1: 1: negative negative control control (no(no inoculation); inoculation); 2: 2: wheat wheat sample sample artificially artificially inoculated inoculated with withFusarium Fusariumspp. spp.

SinceSince wheat wheat grains grains could could easily easily bebe contaminated contaminated withwith aa varietyvariety ofof mycobiotamycobiota beforebefore harvest,harvest, andand during during postharvest postharvest handling handling and and storage, storage, multiplex multiplex PCR PCR assays assays were were applied; applied; a total a of total 34 wheat of 34 grainwheat samples grain samples collected collected from eight from storage eight warehouses storage warehouses were tested were for tested the presence for the ofpresence genes involved of genes ininvolved the biosynthesis in the biosynthesis of mycotoxins. of Themycotoxins. samples wereThe examinedsamples were directly examined by using directly this method, by using without this amethod, fungus isolation without anda fungus incubation isolation step. and Among incubation the 34 grainstep. samplesAmong 22the showed 34 grain positive samples signals 22 showed when mycotoxinpositive signals gene-based when primermycotoxin sets gene were‐based used (Table primer3). sets were used (Table 3).

TableTable 3. 3. Detection of of mycotoxin mycotoxin biosynthetic biosynthetic pathway pathway genes genes by bymultiplex multiplex PCR PCR in stored in stored wheat wheat grain grainsamples. samples. Mycotoxin Biosynthetic Gene Specific Primer Sets Wheat Sample # Aflatoxins (Set No.Mycotoxin VI) BiosyntheticFusarium GeneToxins Specific (Set Primer No. VII) Sets OTA (Set No. VIII) Wheat Sample # aflR1Aflatoxins nor1 (Set No.avfA VI) ver1 fum1Fusarium fum13Toxins tri5 (Set No.tri6 VII) pks13 OTApks (Set No.otanps VIII) 1–3 aflR1 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ nor1 avfA ver1 fum1 fum13 tri5 tri6 pks13 pks otanps 1–34 -+ ‐ ‐ - - -+ -+ -+ ‐ ‐ ‐ ‐ ‐ - - - - - 45 +--+++----+ ‐ ‐ + + + ‐ ‐ ‐ ‐ ‐ - 5 +--+++---- - 6 + ‐ ‐ + + + ‐ + ‐ ‐ ‐ 6 +--+++-+-- - 77 ‐ ‐ ‐ ---+------+ ‐ ‐ ‐ ‐ ‐ ‐ ‐ - 88 ‐ ‐ ‐ ---+------+ ‐ ‐ ‐ ‐ ‐ ‐ ‐ - 99 ‐ ‐ ‐ ---+------+ ‐ ‐ ‐ ‐ ‐ ‐ ‐ - 10 ------10 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ 11 - - - - + + - - - - - 12–1511 ‐ ‐ ‐ ‐ - - - - -+ -+ ‐ ‐ ‐ ‐ ‐ - - - - - 12–1516 - ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ - - + + + - - - - - 1716 ‐ ‐ ‐ - - - -+ -+ -+ ‐ ‐ ‐ ‐ ‐ - - - - - 18 + - - + + + - - - - - 17 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ 19 + - - + + + - - - + - 2018 ++ ‐ ‐ - - ++ ++ ++ ‐ ‐ ‐ ‐ ‐ - - - - - 2119 ++ ‐ ‐ - - ++ ++ ++ ‐ ‐ ‐ - - -+ ‐ - - 2220 -+ ‐ ‐ - - -+ -+ -+ ‐ ‐ ‐ ‐ ‐ - + - + - 23 ------+ - 2421 -+ ‐ ‐ - - -+ -+ -+ ‐ ‐ ‐ ‐ ‐ - + - + - 2522 ‐ ‐ ‐ ‐ ‐ ‐ ‐ - - - + - - -+ - ‐ -+ ‐ - - 2623 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ + - - + + + - - -+ ‐ - - 27–3424 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ------+ - ‐ -+ ‐ - - 25 ‐ ‐ ‐ “+” detected;+ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ”-“ not detected 26 + ‐ ‐ + + + ‐ ‐ ‐ ‐ ‐ 27–34 ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ “+” detected; ”‐“ not detected

Toxins 2017, 9, 302 7 of 17

Figure4 shows some of the results obtained when a multiplex PCR was applied to DNA extracted from wheat samples collected from two storage warehouses in northern Israel; in this case amplification products were obtained, indicating the presence of potentially aflatoxigenic Aspergillus isolatesToxins 2017 (Figure, 9, 302 4A) and toxigenic Fusarium spp. (Figure4B). There was no amplification in7 the of 16 clean, uncontaminated sample that was used as a negative control. As noted above, relatively moderateamounts amountsof fungal ofcontamination fungal contamination (at least 10 (at4 spores/g) least 104 spores/g)could be detected could be by detected multiplex by multiplex PCR. Yet, PCR.theseYet, results these suggest results thatsuggest this method that this can method be used can as be a used diagnostic as a diagnostic tool, which tool, may which enable may the enable rapid theand rapid cost‐ andeffective cost-effective detection detection of potential of potential mycotoxigenic mycotoxigenic fungi in stored fungi in wheat stored grain. wheat grain.

FigureFigure 4. 4. MultiplexMultiplex PCR using DNA isolated from from stored stored wheat wheat grain. grain. (A (A) )Primer Primer set set VI. VI. Lanes: Lanes: 1: 1:negative negative control; control; 2–6: 2–6: wheat wheat grain grain naturally naturally contaminated contaminated by aflatoxicgenic by aﬂatoxicgenic AspergillusAspergillus spp.; andspp.; (B) andprimer (B) primerset VII. set Lanes: VII. Lanes: M: 100 M: 100bp DNA bp DNA ladder; ladder; 1: 1:negative negative control; control; 2–8: 2–8: wheat graingrain naturallynaturally contaminatedcontaminated by by toxicgenic toxicgenicFusarium Fusariumspp. spp.

2.3.2.3. Development Development of of the the Analytical Analytical Method Method

TheThe LC/MS/MS LC/MS/MS multi multi-toxin‐toxin method was optimized forfor thethe detectiondetection andand quantificationquantification of of AFB AFB11,, AFBAFB22,, AFGAFG11,, AFGAFG22, OTA, ZEN,ZEN, DON,DON, FBFB11,, FBFB22, and T-2T‐2 toxins.toxins. Due toto thethe largelarge numbernumber andand thethe chemicalchemical diversity diversity of of the the analytes, analytes, the applicationthe application of an appropriateof an appropriate combination combination of solvents of issolvents required is forrequired extraction for procedureextraction during procedure the development during the ofdevelopment a multi-toxin of method. a multi Different‐toxin method. mixtures Different of water andmixtures organic of solvents water and (such organic as methanol solvents and (such acetonitrile) as methanol with and or without acetonitrile) the addition with or of without acetic acid the wereaddition tested. of Theacetic best acid recovery were tested. and specificity The best values recovery were and obtained specificity using values a water/methanol were obtained (25:75 usingv/v )a mixturewater/methanol for the extraction (25:75 v of/v) the mixture 10 mycotoxins for the fromextraction wheat of grain the samples.10 mycotoxins In contrast from to thewheat majority grain ofsamples. the studies In contrast that have to described the majority an additional of the studies clean-up that procedure have described [34–37], inan the additional current study clean the‐up developedprocedure multi-toxin [34–37], in methodthe current allows study direct the injection developed of the multi crude‐toxin extracts method to LC/MS/MSallows direct system injection with of nothe further crude extracts clean-up to and LC/MS/MS with sufficient system sensitivity with no further and selectivity clean‐up and to detect with andsufficient accurately sensitivity quantify and mycotoxinselectivity contentsto detect in and wheat accurately grains atquantify levels lower mycotoxin than the contents maximum in wheat residue grains limit at (MRL, levels value lower fixed than bythe the maximum legislation). residue The limit mycotoxins (MRL, value of interest fixed inby thisthe legislation). study belong The to mycotoxins different groups of interest in terms in this of chemicalstudy belong properties. to different Therefore, groups the mobilein terms phase of chemical composition properties. was also Therefore, an important the mobile factor which phase influencedcomposition the was signal also intensity an important and, thus, factor the sensitivitywhich influenced of each compound. the signal Severalintensity combinations and, thus, the of mobilesensitivity phase of were each testedcompound. including Several formic combinations acid, acetic acid,of mobile ammonium phase were formate, tested ammonium including acetate, formic methanol,acid, acetic and acid, acetonitrile. ammonium Finally, formate, ammonium ammonium acetate acetate, acidified methanol, with acetic and acidacetonitrile. in combination Finally, withammonium methanol acetate was chosen acidified as thewith best acetic compromise acid in combination for multi-mycotoxin with methanol analyses. was Thechosen gradient as the rate best wascompromise optimized for to multi avoid‐mycotoxin co-eluting compoundsanalyses. The from gradient the matrix rate was (especially optimized for AFGto avoid2 qualifier co‐eluting ion). Mycotoxincompounds validation from the resultsmatrix fulfilled(especially the for acceptance AFG2 qualifier criteria ion). in terms Mycotoxin of accuracy, validation linearity, results selectivity, fulfilled andthe precision.acceptance The criteria calibration in terms levels, of calculated accuracy, concentration linearity, selectivity, (for accuracy), and precision. precision (RSD%), The calibration limit of quantificationlevels, calculated (LOQ) concentration and limit of (for detection accuracy), (LOD) precision are summarized (RSD%), in limit Table of4 .quantification Calibration curves (LOQ) were and linearlimit (R2of detection > 0.995) for(LOD) all compounds are summarized and the in selectivity Table 4. Calibration was acceptable curves for were all compounds linear (R2 (interfering > 0.995) for peaksall compounds were <30% and of LOQ).the selectivity was acceptable for all compounds (interfering peaks were <30% of LOQ).

Toxins 2017, 9, 302 8 of 17

Table 4. Summary of LC/MS/MS validation results.

Compound Precursor Ion m/z Quantiﬁer (Qualiﬁer) Ions m/z Calibration Level (ppb) Calculated Concentration ppb (±SD) 1 RSD (%) 2 LOQ (LOD) ppb 40 41 (3.6) 8.9 355 11.4 DON 295 (265) 80 82.5 (4.8) 5.9 [M + CH COO]− (3.2) 3 640 634.6 (27.4) 4.4 20 21.8 (4.9) 22.5 317 6.3 ZEN 131 (175) 40 36.7 (2.4) 6.4 [M − H]− (1.6) 320 300 (22.5) 7.5 40 37.6 (5.9) 15.7 484 8.1 T-2 215 (185) 80 77.5 (8.8) 11.4 [M + NH ]+ (2.4) 4 640 650.2 (18.9) 2.9 2 2.1 (0.2) 10.2 404 0.8 OTA 239 (102) 4 4.3 (0.25) 5.7 [M + H]+ (0.2) 32 32.8 (2.95) 9.2 20 20.2 (0.9) 4.2 722 8.4 FB 334 (352) 40 41.1 (1.5) 3.6 1 [M + H]+ (2.7) 320 330.4 (27.1) 8.2 20 18.3 (4.0) 21.7 706 6.8 FB 336 (318) 40 38.8 (5.2) 13.5 2 [M + H]+ (2.2) 320 338.4 (23.7) 7.0 1 0.98 (0.1) 10.4 313 0.5 AFB 285 (128) 2 2.04 (0.16) 7.8 1 [M + H]+ (0.1) 16 16.3 (0.39) 2.4 1 1.02 (0.08) 7.6 315 0.4 AFB 287 (259) 2 2.07 (0.11) 5.0 2 [M + H]+ (0.1) 16 15.8 (0.79) 5.1 1 1.04 (0.12) 11.2 329 0.5 AFG 243 (200) 2 2.05 (0.13) 6.2 1 [M + H]+ (0.2) 16 15.9 (1.01) 6.4 1 1.1 (0.10) 9.5 331 1.0 AFG 313 (245) 2 2.12 (0.11) 5.3 2 [M + H]+ (0.4) 16 15.7 (0.4) 2.5 1 Calculated concentrations (±SD) for each calibration level represent the accuracy of measurements; 2 and the relative standard deviation (RSD) represents the repeatability. Toxins 2017, 9, 302 9 of 17

2.4. Mycotoxin Detection in Stored Wheat Grain The results of the multiplex PCR assays were found to be in good agreement with those obtained by chemical analysis of the samples (Table5), which were subjected to mycotoxin screening by LC/MS/MS. The LODs values obtained in the present study ranged from 0.1 ppb for aflatoxins to 3.2 ppb for DON. Overall, 17 wheat samples were positive for mycotoxins above their LODs; however, only six analysed samples were contaminated over the EU regulatory limits with at least one mycotoxin [38]. AFB1 was detected in four postharvest wheat samples at levels above the EU regulatory limit of 2 ppb in grains for human consumption (Table5); these samples were found to be contaminated with A. flavus possessing aflatoxin biosynthesis genes (Figure2A) and A. flavus-specific aspergillopepsin pepO gene (Figure S1A).

Table 5. Mycotoxin contamination in stored wheat grain samples (ppb).

Sample # AFB1 AFB2 AFG1 AFG2 FB1 FB2 DON OTA T-2 ZEN 1–3 ------4 2.1 - - - 179.4 13.3 16.7 - - - 5 - - - - 6.2 2.2 - - - - 6 - - - - 428.4 24.5 1.2 - - 0.7 7 - - - - 4.3 - - - - - 8------9 - - - 0.2 - - 2.8 - - - 10 ------11 ------4.6 - - - 12–17 ------18 - - - 1.1 2340.7 87.9 - - - - 19 - - - 0.3 53.9 4 - - - - 20 6.2 - - 0.4 62.6 4.8 - - - 8.9 21 5.2 - - 0.6 135.1 11.8 - 1.9 - 3.8 22 ------263.5 - - 7.6 23 ------24 ------1746.5 - - 64.8 25 ------26 32.3 1.8 - - 106.7 7.5 - - - - 27 ------28 ------8.1 - - 29 ------22.8 - - 30 ------41.5 - - 31 ------5.7 - - - 32–34 ------"-" not detected.

Molecular analysis of the grain samples revealed amplification of the ver1 and/or aflR1 genes (Figure4A), which are associated with aflatoxin biosynthesis. A PCR-based assay of Fusarium mycotoxin biosynthesis genes showed the amplification of fum1 and fum13 genes associated with fumonisin biosynthesis in four wheat samples (Figure4B, lanes 2–5; samples #18–21). Among the strains isolated from the samples F. verticillioides was identified by morphological and molecular characterization as the predominant species, harbouring the fum1 and fum13 genes (Figure2B). The LC/MS/MS analysis clearly indicated the presence of FB1 and FB2 toxins in the samples with high FB1 contamination (2.34 ppm) in stored grain sample #18 (Table5). Another two wheat DNA samples (#22 and #24) showed amplification of tri6 gene involved in trichothecene biosynthesis, according to the results of the multiplex PCR assay (Figure4B; lanes 6 and 8). When these DNA samples were tested against Fusarium species-specific sets of primers the 570-bp amplification fragment was obtained, which indicated the presence of F. culmorum (Figure S1B). The grain samples were cultured to confirm this identification and were found to be contaminated predominantly with Toxins 2017, 9, 302 10 of 17

F. culmorum that exhibited DON-producing abilities. Type B trichothecene DON, also known as vomitoxin, was detected and quantified by LC/MS/MS in both samples; its concentration exceeded the regulatory limit in sample #24 (1.74 ppm; Table5). It has been reported previously that F. verticillioides and F. culmorum were isolated from various corn growing areas in Israel [39,40]; because of the rise in average temperatures caused by climate change, these species are now frequently reported as the main agents of Fusarium diseases of cereals in the Mediterranean region, especially in wet years [41–44]. Moreover, F. verticillioides and F. culmorum are also known as postharvest pathogens, especially on freshly-harvested grain that has not been dried or stored properly [7,45]. Further research is needed to study the relationship between interacting environmental stress factors (such as temperature and water availability) and the activity of key mycotoxigenic fungi, occurring at postharvest stage of wheat grains, for better understanding the conditions which represent higher risks from mycotoxin production. A strong correlation was found between LC/MS/MS analysis of three stored grain samples (#4–#6), in which FB1 and FB2 were present at values below the regulatory guidelines (Table5), and an established multiplex PCR assay of DNAs extracted from the same wheat samples, resulted in specific amplification of target genes fum1 and fum13. However, some differences were observed between molecular and analytical chemistry results: in particular, samples #18–#21, in addition to fum1 and fum13 fragments, showed amplification of the tri6 gene by multiplex PCR (Figure4B), whereas the production of only fumonisins was observed by multi-mycotoxin LC/MS analysis. This could be because a lack of proper environmental conditions might have inhibited the expression of specific toxin metabolic pathway genes [11]. Furthermore, the PCR assay might yield false positive results because of difficulties in detecting mutations outside the primers’ targeted gene sequence region [46]. Nevertheless, some researchers have pointed out that, for diagnostic purposes of screening food and feed samples, false positive results in the detection of mycotoxigenic fungi are more acceptable than false negatives [10,47]. The use of internal amplification controls can prevent false negatives that might be caused by PCR inhibitors; these controls are often used to rule out failure of amplification in cases where the target sequence is not detected [48]. In the present study the nucleic acid amplification tests did not include internal controls because of the high efficacy of the assays and the positive correlation between the multiplex PCR and LC/MS/MS results (correlation coefficient of 0.99). The need to use internal controls should be determined on a case-by-case basis, because development and implementation of such controls can be difficult, and they can impair assay sensitivity [49]. Moreover, if the reaction failure rate is found to be ≤2% during test verification, routine use of an internal control is not necessary [50]. In conclusion, a relatively small number of stored wheat grain samples were found to be contaminated, mainly by Fusarium and Aspergillus spp. Co-occurrence of different mycotoxins produced by these fungi was observed, although most of the toxins were detected at very low levels (Table5), as mandated by international regulatory standards. Nevertheless, the potential presence of opportunistic human pathogens, such as A. fumigatus, in stored wheat grain may represent a high risk of lung infection (pulmonary aspergillosis) to farm workers who have a compromised immune system. The current study might have important implications for keeping mycotoxin-producing fungi and mycotoxins out of the food-supply chain and reducing the hazard to human health. The strong correlation between multiplex PCR and LC/MS/MS results obtained in the present study demonstrated a rapid, accurate, and sensitive means for detection of mycotoxigenic species and mycotoxins in wheat grains. Combinations of molecular and analytical procedures in food safety laboratories might serve as a reliable diagnostic means for the rapid screening of large numbers of samples. In addition, such an approach could be very useful for obtaining information about the potential toxigenicity of fungal species and their concomitant mycotoxins that might contaminate wide ranges of agricultural and food products. Toxins 2017, 9, 302 11 of 17

3. Materials and Methods

3.1. Strains and Media Standard fungal strains (Table S2) were obtained from the USDA Agricultural Research Service Culture Collection (Northern Regional Research Laboratory, Peoria, IL, USA); they were stored in 25% glycerol at −80 ◦C until use and then were maintained on PD (0.4% potato starch, 2% dextrose) agar plates at 30 ◦C. The strains were grown in YPD liquid medium (1% yeast extract, 2% peptone, 2% dextrose) for genomic DNA extraction.

3.2. Wheat Grain Samples Samples were taken from eight wheat grain storage warehouses in various parts of Israel, 3–6 months after harvest. In each warehouse 1-kg aliquots of grain, destined for human consumption, were collected from the front face and the centre, at points located 1 m in horizontal depth within the grain mass, and from areas close to the walls. Grain temperature and moisture content were in the ranges of 27–33 ◦C and 10.5–12.9%, respectively. The collected samples were kept in sterile plastic bags during transport to the laboratory and on the same day a 100-g aliquot from each thoroughly-mixed sample was frozen in liquid nitrogen, lyophilized, and milled into a ﬁne powder with a grain grinder. The samples were stored at 4 ◦C pending analysis.

3.3. Reagents and Solvents All common chemicals and solvents used for DNA extraction were purchased from Sigma-Aldrich and Bio-Lab (Jerusalem, Israel), respectively, unless otherwise specified. Mycotoxin standards were purchased from Fermentek (Jerusalem, Israel). Methanol and acetonitrile (both LC gradient grade) were obtained from J.T. Baker (Deventer, The Netherlands); ammonium acetate and acetic acid (MS grade) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was obtained using a Micropure purification system (Thermo Scientific, Langenselbold, Germany).

3.4. DNA Extraction Fungal genomic DNA was isolated from lyophilized mycelial mats grown overnight in YPD medium. The DNA was extracted using a lysis buffer containing hexadecyltrimethylammonium bromide (CTAB). Lyophilized mycelial mats were pulverized with 5 mL of 3-mm-diameter glass beads in a disposable 50-mL conical centrifuge tube. Ten millilitres of DNA extraction buffer (l.0 M Tris/HCl, pH 7.5; 1% (w/v) CTAB; 5 M NaCl; 0.5 M EDTA; 1% (v/v) 2-mercaptoethanol; and proteinase K at 0.3 mg/mL) were added to the powdered mycelia and mixed gently, and the mixture was incubated at 65 ◦C for 30 min. The extracts were cooled prior to the addition of an equal volume of chloroform, gently mixed and centrifuged at 6000 rpm for 10 min. The aqueous supernatant was recovered and the nucleic acids were precipitated with an equal volume of 2-propanol. Gentle mixing resulted in the formation of high-molecular-mass DNA, which was precipitated by centrifugation at 4800× g for 5 min. The DNA was resuspended in TE buffer solution (Tris-EDTA, pH 8.0) containing RNase A at 10 µg/mL, and further puriﬁed by phenol-chloroform extraction (A260/A280 ratio of 1.8–2.0). Finally, the DNA was precipitated with 100% ethanol containing 3 M sodium acetate, rinsed in 70% (v/v) ethanol and resuspended in TE buffer. The DNA extraction from wheat grain samples was performed as described above. The purity of the extracted DNA was assayed with an ND-2000 spectrophotometer (Thermo Scientiﬁc, Wilmington, DE, USA).

3.5. Multiplex PCR In order to optimize the multiplex PCR assay for direct detection of mycotoxigenic fungal species in naturally infected wheat grain samples, primers used previously for species-specific detection Toxins 2017, 9, 302 12 of 17 and for amplification of genes involved in mycotoxin biosynthesis (Table1, Table2 and Table S2) were tested for referenced fungi and confirmed by performing monoplex PCR. The multiplex PCR was standardized by empirically varying critical factors that affect multiplexing, such as primer concentrations, template amount, and annealing temperature. Eight different sets of 4–6 pairs of primers were combined for multiplex PCR, with annealing temperature and amplicon size taken into account. Multiplex PCR was performed in a 25-µL reaction mix containing 20–50 ng of each genomic DNA as a template, 2 × multiplex PCR master-mix with 3 mM MgCl2 (QIAGEN, Hilden, Germany) and each primer at 0.2 µM. The PCR sequence comprised: initial heat activation of DNA polymerase at 95 ◦C for 15 min, followed by 35 cycles of denaturation at 94 ◦C for 30 s, annealing at 55–60 ◦C (Tables1 and2) for 90 s, extension at 72 ◦C for 90 s, and a final extension at 72 ◦C for 10 min. PCR products were electrophoresed on a 1% agarose gel with a 100-bp DNA size marker at 96 V for 1 h.

3.6. Multiplex PCR with Artiﬁcially Contaminated Wheat Grain Samples Validation and sensitivity assessment of the method were carried out by inoculation of 2 g of sterile wheat grain samples with the individual spore suspensions (104–106 spores/g) of A. carbonarius, A. fumigatus, and A. parasiticus; an uninoculated sample was used as a negative control. DNA was isolated from the samples as described above, and subjected to multiplex PCR assay.

3.7. Isolation of Fungal Species from Wheat Grain The wheat samples were analysed for presence of potentially mycotoxigenic fungi. The fungi were obtained by direct plating of wheat seeds from each sample onto PDA media supplemented with chloramphenicol at 10 µg/mL, incubated at 28 ◦C for three days, and identiﬁed by morphological analysis and sequencing of ribosomal DNA internal transcribed spacers (ITS). The ITS regions 1 and 2 of ribosomal DNA were used to compare the ITS1-ITS2 nucleotide sequences. The universal ITS primers, ITS1 (50-TCCGTAGGTGAACCTGCGG-30) and ITS4 (50-TCCTCCGCTTATTGATATGC-30) were used and the ITS regions were ampliﬁed selectively by PCR. The sequence of nucleotide alignments obtained was referenced against the GeneBank database [51] with the nucleotide BLAST program.

3.8. Preparation of Mycotoxin Standard Solutions

Individual stock standard solutions (1 mg/mL) of aﬂatoxins B1,B2,G1, and G2 (AFB1, AFB2, AFG1, AFG2), OTA, ZEN, DON, fumonisins B1 and B2 (FB1, FB2), and T-2 toxin (T-2) were prepared in methanol. Multi-toxin working standard solutions with a series of toxin concentrations were prepared by dilution of the stock solutions of the analytes in methanol. All solutions were stored at –20 ◦C and were brought to room temperature before use.

3.9. Sample Preparation for Mycotoxin Analysis Five grams of the ground sample material were mixed with 20 mL of 25:75 (v/v) water/methanol and placed in an orbital shaker for 30 min. After centrifugation at 8500× g for 15 min, 5 mL of the supernatant were transferred to a 15-mL glass tube and evaporated under a stream of nitrogen gas at 50 ◦C. The dry residue was reconstituted with 0.25 mL of a 95:5 (v/v) water/methanol mixture and centrifuged for 10 min at 17,000× g, at 4 ◦C; the supernatant was used directly for the analysis. Samples, in which the concentration exceeded the highest level of calibration, were diluted and re-injected.

3.10. Instrumentation for Mycotoxin Analysis Detection and quantification of mycotoxins were performed with high-performance liquid chromatography coupled with tandem mass-spectrometry (LC/MS/MS). Chromatographic separation was carried out using Nexera X2 UHPLC (Shimadzu, Tokyo, Japan) equipped with 100 × 2.1 mm, ◦ 2.6 µm Kinetex C18 column, (Phenomenex, Torrance, CA, USA). The column was maintained at 40 C and the injection volume was 2 µL. The mobile phase consisted of 2.5 mM ammonium acetate acidified Toxins 2017, 9, 302 13 of 17 with 0.1% acetic acid (A), and methanol (B). The methanol (B) concentration was raised gradually from 5% to 95% within 8 min, brought back to the initial conditions at 9 min, and allowed to stabilize for 3 min. The mobile phase was delivered at a flow rate of 0.4 mL/min. The LC system was coupled with API 6500 hybrid triple quadrupole/linear ion trap mass spectrometer (Sciex, Concord, ON, Canada), equipped with a turbo-ion electrospray (ESI) ion source. The mass-spectrometer was operated in scheduled multiple reaction monitoring (sMRM) in both positive and negative mode within a single run. Positive polarity was applied for all analytes except for DON and ZEA. Precursor/quantifier/qualifier ions are specified in Table4. Source temperature was set at 350 ◦C, ion-spray voltages at −4500 V (negative mode) and 5000 V (positive mode), curtain gas at 35 arbitrary units (au), nebulizer gas at 60 au, and turbo gas at 40 au.

3.11. Validation of Analytical Parameters Validation of the method was carried out in wheat matrix, according to Commission Regulation (EC) No 401/2006 [52]. Three non-contaminated wheat grain samples were spiked with multi-mycotoxin standard solutions at three concentration levels. The calibration levels are speciﬁed in Table4. Extraction and analysis were performed as described above. The spiking experiments were performed in triplicate at three different time points. Validation parameters, such as precision, accuracy, LOD, LOQ, and speciﬁcity, were determined.

Supplementary Materials: The following are available online at www.mdpi.com/2072-6651/9/10/302/s1, Figure S1: Species specific multiplex PCR assays using DNA isolated from stored wheat grain, Table S1: Primer sets used in this study (including references [53–72]), Table S2: Fungal isolates used in this study. Acknowledgments: This study was supported by a grant (no. 20-14-0021) from the Chief Scientist of the Israeli Ministry of Agriculture and Rural Development. Author Contributions: S.S., M.B., and E.S. conceived and designed the experiments; S.S., M.B., V.Z., M.K., A.T., and E.Q. performed the experiments; S.S, M.B., M.K., and E.S. analysed the data; and S.S., M.B., and E.S. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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